Electromigration injection from a microreservoir-electrode in capillary separation systems

ABSTRACT

A capillary electrophoresis system (10) comprising: a separation capillary (20) with a first distal tip (30) and a second distal tip (140); a source vessel (50) containing a solution (40); a microreservoir-electrode (59) comprising a wire loop; a power source (60) connected to the microreservoir-electrode by wire (57); a control system (200); a detector (90); and a final destination vessel (160) containing electrolyte (150) and a ground electrode (155).

RELATIONSHIP TO OTHER PATENT APPLICATIONS

This application is the national stage of International application No.PCT/US97/13663, which claims the benefit of U.S. provisional patentapplication Ser. No. 60/023,074, filed on Aug. 2, 1996 and entitledELECTROMIGRATION INJECTION FROM A SMALL LOOP IN CAPILLARYELECTROPHORESIS.

BACKGROUND OF THE INVENTION

Capillary electrophoresis ("CE") and associated capillary scaletechnologies provide very important analytic techniques for separationand quantitation of large biomolecules. Although such techniques areuseful in separating and detecting small ions, ion chromatography hasbeen a more dominant technique. The more successful ion chromatographydetection techniques have recently been found to be applicable tocapillary electrophoresis. One result has been so-called suppressedconductometric capillary electrophoresis separation systems ("SuCCESS").SuCCESS technology can produce low μg/L limits of detection for avariety of small ions in a robust manner without special efforts towardspre-concentration. (See U.S. Pat. Nos. 5,358,612 and 5,433,838 toDasgupta and Bao.)

However, capillary electrophoresis is most commonly carried out usingUV-Vis absorptiometric detection, as shown in FIG. 1. (See for example,"Capillary Electrophoresis" by S. F. Y. Li, Elsevier, N.Y. 1992. Asshown in FIG. 1, a CE analysis system 10 includes a separation capillary20 whose distal tip 30 initially is in fluid communication with asolution 40 containing analyte samples A, and typically also containingother substances X. Solution 40 is retained in a source vessel 50 and iselectrically coupled by an electrode 55 by a wire 57 to a power source60 that is at a high voltage ("HV") potential V1, typically manykilovolts. A second or ground electrode 155 is often disposed in a finaldestination vessel 160. As shown in FIG. 1, capillary 20 passes througha UV-visible absorption detector 90 before reaching final destinationvessel 160.

Coupling HV power supply 60 to capillary 20 as shown in FIG. 1 resultsin a left-to-right direction migration of analyte A within thecapillary, as indicated by the rightward-pointing arrows. Such migrationcan commence within seconds of energizing power supply 60. Power supply60 may then be turned-off, after which tip 30 of capillary 20 may berelocated into a second vessel 70 containing running electrolyte 80.Power supply 60 is coupled to solution 80 via an electrode 55, which maybe identical to (or indeed the same as) electrode 55 described inconjunction with vessel 50. Power source 60 may then be re-energized,which continues the downstream migration of the sample analyte. Thistype of electric field induced analyte injection is termedelectromigrative or electrokinetic injection ("EI").

The distal end of capillary 140 is in fluid communication withelectrolyte 150 contained in a terminating electrolyte reservoir 150.Preferably electrolyte 150 is the same as running electrolyte 80, and inthe embodiment shown is at ground potential.

As noted, during EI, HV is applied with the background electrolyte(BGE)-filled capillary dipped in a sample vial. In a typical situation,electroosmotic and electrophoretic movements act together to introducethe desired class of analyte ion(s) into the capillary. In general, whenthe electroosmotic mobility (μ_(eo)) is small relative to theelectrophoretic mobility (μ_(eo)), conditions are most favorable forelectromigrative preconcentration. Under these conditions a significantamount of analyte can be introduced without the concomitant introductionof a significant liquid volume. EI has been widely used for the traceanalysis. This is especially valuable with UV-Vis detection becauseon-column UV-Vis absorption detectors, e.g., detector 90, typically usedin CE provide relatively poor concentration detection limits. In thedetermination of small ions, where indirect detection is typically used,the situation is even less favorable than with direct detection.

In EI, when the sample ionic strength is very low, best results areobtained if a low mobility ion is deliberately added to the sample at aconcentration that is high relative to the total concentration of theanalyte. By "low mobility" what is meant is an ion having mobility lowerthan any of the analyte ions of interest. In such case, the added ionbehaves like a terminating electrolyte and electromigrativepreconcentration closely resembles isotachophoresis. In some situations,a high mobility ion is the analyte of interest and low mobility ions arealready present in abundance, such as in the determination of residualsulfate in sulfonate dyes. There is no need to add any terminatingelectrolytes in such cases. In cases where the analyte of interest ispresent at a low concentration in a sample that has a significant ionicstrength, it is impractical to add sufficient terminating electrolyte tomake the latter the dominant current carrier.

Unfortunately, even under identical sample analyte concentrations andinstrumental settings, the amount of an analyte introduced into an EIsystem is a strong function of sample conductance. This relationshipoccurs because conductance affects the rate of electroosmoticintroduction. Less directly conductance also affects the rate of theelectrophoretic movement through a change in the field strengthexperienced by the sample. Further, EI is dependent on the mobility ofthe analyte itself, creating a bias in favor of the high mobility ions.By "bias" it is meant that the injected sample differs from the originalsample. The difference occurs because there is a relative deficit ofslower moving ions, and a relative excess of faster moving ions in thealiquot injected portion as contrasted to what was originally present inthe sample. Although researchers such as Lee and Yeung, Anal. Chem.1992, 64, 1226-1231, have advanced a simple approach to improveprecision of the results obtained in EI through monitoring systemcurrent, the Lee-Yeung technique does little to solve the problem causedby biased injection.

Other attempts have been made in the prior art to address theabove-noted bias dependency of sample conductivity. For example, the useof two separate internal standards that bracket the entire range ofanalyte mobilities of interest has been suggested, as has been thestandard addition of every analyte of interest. These approaches areunsatisfactory and indeed can be tedious.

Finally, the prior art has tended to overlook the fundamental fact thatthe EI of sample ions into a capillary is ultimately dependent upon thelocal electrical field. Any changes in the geometry and or the physicaldistance between the HV electrode and the capillary tip can profoundlyaffect EI. Unfortunately, it has been difficult in the prior art toreliably produce a truly symmetrical electric field.

In the prior art, the total amount of analytes in the sample aliquotfrom which analyte ions are EI-introduced into the capillary is verylarge relative to the amount of analytes actually introduced. If onecould perform EI from a truly small sample volume for a long enoughperiod, it would be possible, in principle, to introduce virtually allthe analyte ions of interest into the capillary in an exhaustive manner.The sample volume would not become deionized or become non-conductive inthe process. Deionization or conductivity loss would not occur becauseelectro-generated H⁺ or OH⁻, and the appropriate counter-ion alreadypresent in the sample, and those migrating against the EOF into thesample from the capillary, would maintain the sample conductive. Indeed,if EI could be carried out long enough, significant amounts of H⁺ or OH⁻would be introduced. Unfortunately exhaustive electromigration cannot beeffectively practiced in the prior art.

Thus, there is a need for an easily produced microreservoir, preferablyhaving a sub-μL liquid capacity, that can be used in exhaustiveelectromigration and electrophoresis. Preferably such microreservoirshould permit the entire sample within to be readily subjected to asymmetrical electric field. Further, the microreservoir should permitthe entry tip of a separation capillary to be disposed at thesymmetrical center of the sample. A system incorporating these featuresand methodology would advantageously reduce the effects of conductivityin electromigration injection capillary electrophoresis.

The present invention provides such a method and apparatus.

SUMMARY OF THE INVENTION

Undesired bias effects are reduced in a separation system usingelectromigration injection ("EI") by exhaustively introducing analyteions of a desired polarity in the sample into the separation capillaryusing a microreservoir-electrode. (The separation system may be anelectrophoretic or sn electrochromatographic system.) A wire loop orhemisphere formed in a metallized base defines a microreservoir offinite volume that preferably is symmetrical to reduce the time requiredto achieve exhaustion. The entry tip of the separation capillary ispreferably disposed at the center of the microreservoir, whichadvantageously reduces the time required to achieve exhaustion.

Because the microreservoir is an electrical conductor, it is coupled toone terminal of the high voltage power source and constitutes one of thehigh voltage electrodes in the separation system. With high voltageapplied, the sample under analysis is subjected to an electric fieldwithin the microreservoir, and the separation system is used to conductan exhaustive injection of the analyte. Advantageously, conductingexhaustive electro-injection in the separation system substantiallyreduces bias effects resulting from faster moving ions in the sampleentering the capillary sooner than slower moving ions of the samepolarity. Exhaustive injection using electromigration results inessentially quantitative sample injection. This injection occurssubstantially independently of the conductivity of the sample, andsubstantially independently of the ionic mobility of the various analyteions of the same polarity.

Exhaustive electroinjection is preferably carried out as follows. Usinga preferably symmetrical microreservoir-electrode whose volume isreproducible and small, e.g., <2 μL, a small aliquot or portion istaken. The portion may be a film that is formed by dipping andwithdrawing a loop microreservoir-electrode into and out of a vesselcontaining the sample. Using the microreservoir-electrode as oneelectrode, EI potential is applied for a desired period, e.g., 30seconds to 60 seconds, during which time all ions of the desiredpolarity present in the sample are essentially quantitatively injectedinto the tip of the separation capillary. At first the faster movingions are injected and as these ions are depleted, slower moving ions areinjected into the capillary. This result is attained because thereservoir volume is relatively small. The result is that bias effectsare reduced in that the injected sample will contain a truerrepresentation of the more slowly moving ions, in addition to the fastermoving ions.

Other features and advantages of the invention will appear from thefollowing description in which the preferred embodiments have been setforth in detail, in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a conventional separation system, according to the priorart;

FIG. 2 depicts an EI separation system, according to the presentinvention;

FIG. 3A depicts a first embodiment of a microreservoir-electrode usinginclined loop geometry and an associated support wire, according to thepresent invention;

FIG. 3B depicts a second embodiment of a microreservoir-electrode usingplanar loop geometry in which the capillary longitudinal axis and loopradius are on a common plane, according to the present invention;

FIG. 3C depicts a third embodiment of a microreservoir-electrodecomprising a hemisphere formed in a conductive base material, accordingto the present invention;

FIG. 4 depicts numerical simulation EI data from an well-mixed film,according to the present invention;

FIG. 5 depicts numerical simulation of EI from a film in which no mixingis present, other conditions being the same as for FIG. 4, according tothe present invention;

FIG. 6 depicts spatial distribution of chloride, acetate and hydroxidein a film as a function of EI period, other conditions being the same asfor FIG. 5, according to the present invention;

FIG. 7 depicts numerical simulation of EI from a film in which the modelincludes diffusional mixing, other conditions being the same as for FIG.5, according to the present invention;

FIG. 8 depicts spatial distribution of chloride, acetate and hydroxidein a film as a function of EI period, in which model includesdiffusional mixing, according to the present invention;

FIG. 9 depicts fraction of chloride and acetate injected as a functionof loop radius and EI period, according to the present invention;

FIGS. 10A and 10B depict, respectively, conventional EI and EI from aloop, according to the present invention;

FIG. 11A depicts EI from a small loop exhibiting an extremely largedynamic range with variation of acetate over three orders of magnitudeat constant nitrate concentration, according to the present invention;

FIG. 11B depicts variation of nitrate over 3 orders of magnitude atconstant acetate concentration, according to the present invention;

FIG. 12 depicts loop-EI normalized data-for chloride to acetate peakarea ratio as a function of HV and EI period, according to the presentinvention;

FIG. 13A depicts loop-EI equivalent volume injected as a function of EI,according to the present invention;

FIG. 13B depicts loop-EI bias relative to chloride as a function of EI,according to the present invention; and

FIG. 14 depicts hydroxide introduction upon prolonged EI from a loop,according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 2 shows a somewhat modified CE system 10' in which amicroreservoir-electrode 59 is provided. As will be described herein, amicroreservoir-electrode preferably defines a volume holding capacity ofapproximately 1 μL or less and advantageously serves as an electrode, inlieu of electrode 55 in prior art FIG. 1. The microreservoir-electrodegeometry is symmetrical, preferably a loop formed from conductive wire(see FIGS. 3A, 3B) or a hemisphere defined in a metal or conductive base(see FIG. 3C).

System 10' was used by applicants as a fully automated custom designedCE system that used fused silica capillaries 20 having 74 μm innerdiameter, 360 μm outer diameter, and 60 cm lengths. Such capillaries areavailable from Polymicro Technologies, Phoenix, Ariz. The high voltage("HV") power source 60 was provided by a programmable HV power supply,model CZE 2000, available from Spellman High Voltage, Plainview, N.Y. Avariable wavelength UV-Vis on-column absorbance detector 120 or 130 wasused (a LINEAR model 206 PHD available from Thermo Separation Systems)in combination with UV-206 data acquisition software (LINEAR) operatingon a 80386 class personal computer (PC) (not shown). Of course otherequipment and other sized capillaries might be used. Those skilled inthe art will appreciate that system 10' may in fact comprise a capillaryelectrochromatography system. In such case, capillary 20 would be apacked column rather than a hollow tube.

An automated system that precisely controls withdrawal of the capillarysampling head from the sample is especially important in practicing thepresent invention. It is desired to withdraw a reproducible amount ofliquid from vessel 50 as a film on a wire loop, such asmicroreservoir-electrode loop 59 shown in FIGS. 3A and 3B. A slow rateof withdrawal is needed to ensure a reproducible amount of liquidretained in the film. In applicants' experiments, system automation wasaccomplished by using a modified fraction collector (model 2110,BIO-RAD, Richmond, Calif.) as an autosampler. A number of pneumaticlinear actuators, governed by electrically operated air solenoid valves,served to provide horizontal and vertical motion to the capillary head.System operation was controlled by a programmable microcontroller (aMicro Master LS unit available from Minarik Electric, Los Angeles,Calif.). It is understood that other control systems may instead beemployed. Collectively, these components and sub-systems are showngenerically in FIG. 2 as 200. Physical movement ofmicroreservoir-electrode 59 is not shown to avoid cluttering FIG. 2.

Applicants' publication in Anal. Chem. 1996, 68, 1164-1168 disclosedgeneral techniques to form wire loops at the tip of a capillary. Bycontrast, the loops used in the embodiments of the present inventionshown in FIG. 3A and 3B are substantially larger and are thereforefairly simple to fabricate. Briefly, 5/64 inch to 1/8 inch diameterloops were initially made by wrapping 135 μm diameter stainless steelwire around suitably sized drill bits and were held with one or twotwists.

The wire loop embodiment microreservoir-electrodes (abbreviated "loop")were used in two different geometries, shown in FIGS. 3A and 3B. In FIG.3A, the plane of loop 59 is inclined at an angle φ of 45° to 90°relative to the longitudinal axis of the separation capillary 20. Avertical support wire 64, parallel to capillary 20, holds loop 59 inposition. Note that the loop is symmetrical, and that the center of tip30 is disposed symmetrically at the center of the loop. The support wireand the capillary were retained a few cm above the lower capillary tip30 by a small Plexiglas jig, depicted as 66. A conductive wire 57couples the wire loop to the HV power supply 50, such that loop 59 formsone electrode in the separation system 10'. An advantage of using a loopmicroreservoir-electrode is that a very symmetrical electric field isproduced across the electrode. In the embodiment of FIG. 3A, uponwithdrawal from a liquid, the amount of liquid held in the loopdecreases as the loop plane angle becomes more vertical.

In the embodiment of FIG. 3B, loop 59 is formed around the capillary tip30, the capillary axis now being parallel to the plane of the loop. Theterminal wire 68 is wrapped around the tip of the capillary and isaffixed thereto by epoxy adhesive.

In the embodiments of FIGS. 3A and 3B, high voltage connections weremade to the protruding wire (or support wire) 64 or to a wire 57 coupledto the loop. As noted, the result is that loop 59 serves as a HVelectrode, in lieu of electrode 55 in FIG. 1. Because the presentexperiments were limited to reversed polarity (-HV), stainless steelmaterial was adequate to fabricate loop electrodes 59.

As noted, it is desired that microreservoir-electrode 59 retain a finiteamount of liquid, e.g., <1 μL, and provide a uniform electric fieldacross the microreservoir when used in system 10'. A symmetrical loopsuch as shown in FIGS. 3A and 3B meet these goals. In the embodiment ofFIG. 3C, microreservoir-electrode 59 comprises a base of electricallyconductive material, e.g., metal, having a surface in which a hemispheredepression 68 is formed. Although the base is shown as square, for easeof illustration, a circular base could instead be used, and wouldfurther contribute to symmetry. The hemisphere depression is sized toretain a desired amount of liquid, e.g., <1 μL, thus serving as amicroreservoir of finite volume capacity. For retaining a fixed volume,such a reservoir may also be made to be self-siphoning. Because themicroreservoir is made of an electrically conductive material, wire 57may be connected to the base of microreservoir-electrode 59, permittingit to also function as an electrode. Again, the tip 30 of capillary 20is disposed symmetrically at the center of the microreservoir hemisphere68. The overall result is that a sample retained in hemisphere 68 issubjected to a symmetrical electric field.

With respect to reagents used with system 10', all solutions wereprepared in distilled deionized water (e.g., Barnstead Nanopure) havingspecific resistance greater than 16 MΩ·cm. Sodium chromate (5 mM) wasmade fresh daily from a 50 mM stock prepared from Na₂ CrO₄.4H₂ O, A. R.Grade, Mallinckrodt, adjusted to pH 8.0 with 0.1 M H₂ SO₄.Cetyltrimethylammonium hydroxide (CTAOH) was prepared by ion exchanginga cetyltrimethylammonium chloride solution through an OH⁻ -form 200-400mesh Dowex 1×8 anion exchange column, and used from a 20 mM stock. CTAOHwas used as the electroosmotic flow ("EOF") modifier (to achieve flow tothe ground electrode with -HV applied) and was added to the chromateelectrolyte to attain a final concentration of 0.5 mM.

In operating system 10', capillary 20 was first filled with the BGE andthen tip 30 was lowered into and withdrawn from one sample vial forwashing loop 59. The capillary then entered a second identical samplevial and was slowly withdrawn to form a sample film on the loop. Thecapillary was next moved horizontally and lowered into a position at alevel equal to the liquid level on the destination side, e.g., vessel160 in FIG. 2. In this position, the region around the loop iscylindrically enclosed and a small flow of N₂ (≈20-25 cm³ /min) servesto prevent excessive intrusion of CO₂. Electromigration voltage (-3 kV,except as stated) was then applied from HV source 60. As noted,microreservoir 59 served as one electrode in system 10', the otherelectrode being coupled to electrode 155 (or to some other location insystem 10').

After a desired EI time period, the capillary is put into a first BGEvial for washing and is then lowered into a second, fresh, BGE vial foroperation. An operating voltage of -18 kV was used for electrophoresis.Essentially the same procedure was used in an analogous manner when EIwas carried out from a conventional sample vial. The determination ofanions was studied and except as described, the sample constituted amixture of chloride (200 μg/L), nitrate (400 μg/L), formate (400 μg/L)and acetate (400 μg/L), all as sodium salts.

Consider now the underlying principles at work when practicing thepresent invention. In EI, the quantity Q_(i) of species i introducedduring time t into a capillary of length L and inner radius r_(C),across which a voltage V is applied is given by:

    Q.sub.i =(μ.sub.i +μ.sub.eo)πI.sub.C.sup.2 VC.sub.i t/L(1)

where μ^(i) and μ^(eo) are respectively electrophoretic andelectroosmotic mobilities and C_(i) is the concentration of species i.This analysis assumes that the field E at the tip of the capillary isthe same as the field inside the capillary and is therefore given byV/L. The flux occurs through the capillary inlet cross section. The bulkreservoir of analyte i is so large that C' is essentially unchangedduring the duration of EI. Thus, as long as the rate of migration of theanalyte ion within the capillary does not influence the rate of analyteintroduction at the capillary tip, a more general formulation ofequation (1) will be:

    M.sub.i =dQ.sub.i /dt=(Eμ.sub.i +u.sub.eo)aC.sub.i      (2)

where M_(i) is the mass transport rate (eq/s) of ion i through any areaa around the capillary tip towards the capillary where the concentrationof species i is C_(i).

In practice, the field E inside and outside the capillary is unlikely tobe the same. Nevertheless, the electroosmotically introduced componentof EI is governed by the EOF generated in the capillary. Therefore, theEOF governed component of EI is better specified directly, in terms ofthe bulk flow velocity μ_(eo) generated in the capillary. Obviously, ifμ_(eo) is very small relative to E.sub.μi, equation (2) simplifies to:

    M.sub.i =Eμ.sub.i aC.sub.i                              (3)

Consider now the field geometry at the capillary tip, and filmresistivity. As a representative simplified case, consider that thesample constitutes a thin circular disk of radius r_(out) and thicknessh, and that capillary tip is placed at the center of the disk. As longas h is small relative to r_(out), one may assume that diffusivetransport in the vertical direction is not a limiting process.

The perimeter area of the disk, 2πr_(out) h, constitutes one electrode.Note that even if the film/disk were thicker than the diameter of thewire constituting the loop, the electrode area would still be correctlyapproximated by the entire perimeter area for a thin disk. The capillarycross section, πr_(C) ², represents the second, virtual, electrode. Inmost cases, r_(C) is very small compared to r_(out). Thus, instead of aplanar geometry, one may approximate the central electrode as having acylindrical geometry of radius re with an area equal to the capillarycross section. Whence,

    r.sub.e =r.sub.C.sup.2 /2h                                 (4)

Thus, the result is an annular electrode system, initially having amedium of homogeneous resistivity ρ filling the annulus, where:

    ρ=1/(Σλ.sub.j C.sub.j)                    (5)

in which λ_(j) is the equivalent conductance (in S cm² eq⁻¹) of ion j,and in which C_(j) is its concentration in equivalents/cm³. This yieldsresistivity ρ in Ω·cm. Ions j not only include the analyte anions ofinterest (ions i, e.g., chloride), but also include an equalconcentration of counter-ion (e.g., sodium) that is present in themedium for maintaining electro-neutrality.

In this analysis, applicants have assumed that the rate limiting processof interest is the migration of ions i into the capillary. As such, thereverse migration of the counter-ion from the capillary (where it ispresent in very large concentration relative to concentration of theanalyte ions in the film) is not the limiting step. It should also benoted that EI is accompanied by the electrolytic production of OH⁻ atthe loop electrode that is maintained at a negative potential. Not onlyresistivity ρ in the loop film is affected by an increasing NaOHcontent, but OH⁻ is also thence introduced along with the sample ions,the relative amount increasing with increasing EI period.

Consider the effects of electrical resistance of the loop. If the innerelectrode has a radius r and a thickness h, and if the second electrodeis situated an infinitesimal distance dr away, the inter-electroderesistance dR is given by:

    dR=ρdr/2πrh                                         (6)

Whence

    R=(ρ/2πh)logr                                       (7)

Thus, applying the above to the embodiments of interest in which thereis a loop of outer radius rout and inner radius r_(C), the resistance ofthe loop, R_(loop) is given by:

    R.sub.loop =(ρ/2πh)log(r.sub.out /r.sub.e)          (8)

Mass transfer into the capillary will now be described. In equation (3),the field E may be expressed as:

    E=dV/dr                                                    (9)

Current I flowing through the system is a function of total appliedvoltage V and the sum of the electrical resistance of the capillary(R_(cap)) and the resistance of the loop (R_(loop)). The currentrelationship is as follows:

    I=V/(R.sub.loop +R.sub.cap)                                (10)

During EI, total applied voltage remains constant during EI, and, inmost practical applications, electrical resistance of the capillary(R_(cap)) is essentially invariant and is much greater than R_(loop).current I remains essentially constant. Whence equation (9) becomes:

    E=IdR/dr                                                   (11)

At the tip of the capillary, area a from equation (3), through whicharea the mass transfer occurs, is the surface area of the innercylindrical electrode, and is also equal to the capillary cross section.Area a may be written as:

    A=2πr.sub.e h                                           (12)

Setting r=r_(C) at the capillary tip, equation (6) then yields:

    dR/dr=ρ/2πr.sub.e h                                 (13)

Combining equations 3, 11, 12, and 13 yields the simple result that:

    M.sub.i =Iρμ.sub.i C.sub.i                          (14)

A sample film is the present invention is well mixed spatially and ishomogeneous. Assume that the entire film is well mixed at all times. Forall the anions other than hydroxide,

    -dC.sub.i /dt=M.sub.i /V.sub.f =Iρμ.sub.i C.sub.i /V.sub.f(15)

where V_(f) is the volume of the loop film, given by πr² _(out) ·h.

OH⁻ is also produced in the film at the rate of I/F eq/s, where F isFaraday's constant. It then follows that:

    -dC.sub.OH /dt=I(ρμ.sub.OH C.sub.OH -1/F)/V.sub.f   (16)

Expanding equation (5) indicates that:

    ρ=ΣC.sub.i λ.sub.i +C.sub.OH λ.sub.OH +λ.sub.Na (C.sub.OH +ΣC.sub.i)               (17)

Equations 15, 16, and 17 constitute a set of coupled second orderdifferential equations for which no general solutions exist. However,the system of equations may readily be solved numerically.

Except as stated otherwise, the following system characteristics wereand are assumed: loop radius 1 mm, sample volume 1 μL, samplecomposition Cl (200 μg/L), NO₃ ⁻ (400 μg/L), HCOO⁻ (400 μg/L), CH₃ COO⁻(400 μg/L), EI voltage -3 kV, capillary inner diameter 75 μm, BGE 5 mMNa₂ CrO₄. For a capillary length of 60 cm, computed R_(cap) is 1.008 GΩ,which is in close agreement with an observed current of -3 μA.

Computations were carried out with code written in TURBO BASIC (producedby Borland International) on a Pentium processor based PC operating at133 MHz. Convergence of the solutions was checked by decreasing the timeinterval of iteration steps. Typically no significant changes wereobserved below an iteration step of 100 μs. All of the data describedherein were based on such temporal iteration step.

It should be noted that the computing time for the above well mixed filmcase was not particularly demanding. But, in a more realisticenvironment where film composition changes radially during EI (describedherein), simulating 30 s of EI with a 50 μs iteration step requires acomputing time of over 14 h.

Understandably a 14 h computational time probably represents theacceptable upper time limit for PC-based computations.

Applicants' analysis was according to the following algorithm steps:

1. From the initial sample composition, compute initial value of ρ(equation 17), R_(loop) (equation 8), and I (equation 10);

2. Calculate the mass (cumulative mass) of analyte ions and OH⁻ injectedwithin the chosen iteration time interval (equation 14);

3. Calculate change in composition of the sample film, both in regard toanalyte ions and NaOH (equations 15 and 16), and hence for the newcomposition;

4. Using the new composition, perform the same calculations as in step1, above;

5. Cycle through steps 2, 3, and 4 until the total desired EI period hasbeen simulated.

FIG. 4 shows the results of such a numerical simulation of EI for awell-mixed film. With increasing EI time, the total amount of eachanalyte injected approximately exponentially reaches respective limitingplateau values. However, with increasing EI time, the amount of OH⁻injected (shown with asterisks) increases linearly. Although none of theanalytes is quantitatively injected under such condition, at the end ofthe EI period, fast moving chloride and nitrate are nearly completelyinjected, 96% and 95%, respectively. However, acetate, the slowest ofthe four analytes, is injected to an extent of about 81%. The relativebias of chloride vs. acetate is 1.183, e.g., the extent to which theratio of the injected amounts of chloride to acetate is greater than theratio of the respective ions originally in the sample.

By contrast, when one computes the same relative bias for conventionalEI from a sample vial from equation (1), the value obtained is the ratioof the two mobilities, 1.866. Indeed, applicants experimentally observedthis as well, although data are not shown. Thus, a very significantreduction of bias can be expected if EI is carried out from a finite,limited volume, according to the present invention.

Spatial concentration gradients occur during EI. While the methodologyoutlined above is instructive, it is somewhat over simplifying. Inreality, the loop size is finite. Further, the relatively rapid movementat different rates of ions towards the capillary entrance tend to ensurethat not only the temporal composition but also the spatial compositionof the film will not be constant during the EI process.

During EI, ions are depleted near the capillary entrance and must bereplenished. On the other hand, in the simple model, while OH⁻ isactually produced in substantial quantity near the outer periphery ofthe film, it is assumed to be instantaneously mixed throughout the film,thus lowering calculated film resistance. Consequently the field acrossthe loop and the rate of EI is also lowered, relative to what mightotherwise be expected. While several levels of sophistication may beincluded in a model to account for these occurrences, numericalsolutions are resorted to when equations pertaining to the basic modelcannot be solved analytically.

The basic approach taken by applicants assumes that the film is radiallydivided into a number of thin segments that are individually of uniformcomposition. The innermost zone has a radius of RINZONE. The rest of theloop is divided outward in segments of thickness Δr, until r_(out) isreached. Except as stated,-simulations herein assume RINZONE to be 50μm, which is slightly larger than the capillary inner radius of 37.5 μm.A value ΔR=10 μm is used throughout herein. For r_(out) =0.1 cm, thereare n=96 separate zones. These n zones may be designated in terms oftheir radii in cm, r₁, r₂, . . . r₉₅, r₉₆, where r₁ =RINZONE, r₂ =r₁+0.001, r_(k) =r₁ +(k-1)·0.001, r₉₆ =r_(out), with r₀ designated as 0.Adopting this nomenclature, the volume V_(k) of each zone or shell isgiven by:

    V.sub.k =π(r.sub.k.sup.2 -r.sub.k-1.sup.2)h             (18)

The electrical resistance R_(k) of each shell is given in a manneranalogous to equation (7), namely:

    R.sub.k =(ρ/2πh)log(r.sub.k /r.sub.k-1)             (19)

with the provision that in this case r₀ is not zero but is the radius ofthe hypothetical cylindrical central electrode, r_(C). R_(loop) is thencalculated from equation (20) as: ##EQU1## Equation (10) can now be usedto compute the current. During the initial steps, one can computeamounts of each of the analytes and OH⁻ in each of the zones A_(i),k andA_(OH),k as follows:

    A.sub.i,k =C.sub.i,k V.sub.k                               (21a)

    A.sub.OH,k =C.sub.OH,k V.sub.k                             (21b)

The equation for OH⁻ and for the analytes will generally be the same,and need not be replicated unless there is a difference. Thus, allequations for analytes for which a corresponding equation exists for OH⁻will be designated by `a` after the numeric reference, which willindicate an identical corresponding equation `b`, not set forth herein,that applies to hydroxide. The following method steps are then carriedout:

6. The amount of analyte i transferred from zone k to zone k-1,ΔiA_(i),k over an iteration period Δt is calculated essentially perequation (14):

    ΔA.sub.i,k =Iρ.sub.k μ.sub. C.sub.i,k Δt(22a)

7. The new amounts in each zone are then calculated:

    A.sub.i,k (new)=A.sub.i,k (old)-ΔA.sub.i,k +ΔA.sub.i,k+1(23a)

For all the analytes, at outermost zone n, ΔA_(i),K+1 is taken to bezero. However, hydroxide is generated at this zone and thus:

    ΔA.sub.OH,n+1 =IΔt/F                           (24)

8. The amount ΔA_(i),1 the amount of analyte introduced into thecapillary, a cumulative tally of which is kept.

9. For each zone k, the value of ρ_(k) is computed according to equation17 and the current I is computed using equations 19, 20, and 10.

10. Method steps 6, 7, 8, and 9 are repeated until the desired EI periodhas been reached.

FIG. 5 depicts an EI simulation using the above approach, in which amodel predicted quantitative injection of chloride in 5 s, and predictedeven acetate within 10 s. FIG. 6 depicts radial analyte distributions asa function of EI times. Note in FIG. 6 the radial distribution of theanalytes as EI time progresses. Both chloride and acetate are depletedfrom the outer zones first, and then attain virtual plateaus as theyapproach the center of the loop (or other finitemicroreservoir-electrode). The chloride disappears much faster than theslower moving acetate. OH⁻ is generated at the outer electrode and has adistribution that at any time exhibits a concentration that increasesalmost exponentially as the outer zone is approached. This increaseincreases with increasing EI period, and is essentially the oppositebehavior of the analyte ions. The residual concentration of the analytesin the outermost zone is artificially high, as described herein.

Applicants' model attempts to account for diffusion. At first glance, itmay seem that liquid phase diffusion is slow and may be neglectedrelative to electric field-governed transport of ions. While this may betrue during EI initial stages, diffusive transport has two importanteffects when dealing with nearly exhaustive analyte introduction. First,diffusive mixing may oppose unidirectional electromigration governedtransport, and thereby prolong the time necessary to introduce anyanalyte in a near quantitative fashion. Second, during the latterportion of an EI period in the outermost zone, NaOh concentrationbecomes sufficiently high that the zone is very conductive, and thelocal electric field is low. In this case, diffusion will greatly aidtransport, especially for OH⁻, inward from the outermost zone. One mayassume Fickian diffusive transport between two neighboring segments suchthat the amount of species i transported by diffusion from segment k tosegment k-1, ΔADi,k is given by:

    ΔA.sub.Di,k =D.sub.i (C.sub.i,k -C.sub.i,k-1)a/Δr(25a)

Where D_(i) is the diffusion coefficient (also equal to μ_(i) RT/F), ais the interfacial area of transport given by 2π_(k-1) h, and Δr is thediffusion distance, the mean radial distance between two adjoiningsegments, given by (r_(k) -r_(k-2))/2. R is the universal gas constant,and T denotes absolute temperature. The modified form of equation (22),which takes into account diffusive transport is thus given by:

    ΔA.sub.i,k =μΔt(Iρ.sub.ki C.sub.i,k +4πRTr.sub.k-1 h(C.sub.i,k -C.sub.i,k-1)/(F(r.sub.k -r.sub.k-2)))        (26A)

Iterative computation is then carried out along the same lines asdescribed earlier herein. FIGS. 7 and 8 depict the outputs correspondingto FIGS. 5 and 6, except that diffusion is now taken into account. Notethat consideration of diffusional mixing has a significant effect upondecelerating the rate of analyte introduction through EI. The greatermixing of hydroxide from the outermost zone is also notable. For thesame reason, the artifact residual analyte concentrations in theoutermost zone is no longer visible. The differences between FIGS. 7 and8 and counterpart FIGS. 4 and 5 appear to arise solely from diffusioneffects. The electrical mobility of an analyte is linearly related toits diffusion coefficient. But while electromigration is an electricfield-dependent process, diffusion is not. Consequently, the relativeimportance of diffusion increases at lower applied EI voltages. Thedifferences between the two sets of results can thus be interpreted interms of how changes in the applied EI voltage may affect the process.Consider the effect of changing applied voltage in a numerical model. Incontrast to equation (1), at a constant voltage time product, EI is moreefficient at higher applied voltages. For example, for acetate 94.59%vs. 91.33% is introduced with 5 s at -6 kV, vs. 10 s at -3 kV, and99.87% vs. 99.62% is introduced with 5 s at 18 kV vs. 10 s at 9 kV.

Applicants have also attempted to account for effects fromnon-diffusional mixing and electroosmotic flow. Admittedly, assumingthat the entire film is one well mixed pot is an oversimplification.However, assuming that the only mixing process is diffusion can also beinaccurate.

Applicants' iterative computational procedure assumes that the contentsof every 10 μm thick segment is homogenized every 100 μs, whichassumption represents a process substantially more efficient thandiffusional mixing alone. However, because the computations converge atiteration steps <100 μs, no error appears to be thus incurred. In anreal system, there is reason to believe that more efficient means ofmixing exists, particularly in the innermost and outermost zones. In thefirst case, the presence of the capillary tip may bring aboutnon-diffusional mixing, and in the second case, electrolytic gasevolution may lead to significant mixing.

These phenomena can be accounted for by a choice of radial distancesthat define the innermost and outermost zones because such selectioneffectively changes the volume that is homogenized at each iterationstep. In applicants' model, varying RINZONE between 50 μm and 150 μm(while the outermost zone width and the loop radius are maintainedconstant at 10 μm and 1000 μm, respectively) had very little effect onthe results. The fraction of chloride injected in 10 s decreases from99.4790% to 99.4786% with the stated increase in RINZONE, and the changefor acetate is also very small, from 91.1068% to 91.1009%.

The effect of changing the width of the outermost zone is morepronounced. In this case, the fraction injected of all ions decreases aszone width is increased. RINZONE and the loop radius were maintainedconstant at 50 μm and 1000 μm, respectively, for the outermost zonewidthof 10, 50, 100, 200 and 350 μm. The fractions of chloride injected in 10s were respectively 99.48%, 99.40%, 99.04%, 97.56%, and 94.40%, and thefractions of acetate were 91.11%, 90.82%, 89.72%, 86.01%, and 79.52%.The greater effect of changing the width of the outermost zone isunderstandable. NaOH is produced in this region and there can be largechanges in the effective electric field, which can affect a greatervolume if the changes occur over a larger volume.

Electroosmotic flow, Q_(eo), during EI in the present system is quitesmall, 0.500 nL/s. To account for electroosmotic flow, it is assumedthat the amount introduced by EOF within any given iteration periodcorresponds to the same composition as in the innermost zone. Themodified form of Step 8 (immediately after equation (24)) is thusformulated:

    m.sub.i,inj (new)=m.sub.i,inj (old)+ΔA.sub.i,k +Q.sub.eo ΔtC.sub.t,1                                         (27)

where m_(i),inj is the amount of analyte i injected into the capillaryand ΔA_(i),k is given by equation (26). The volume change in the filmdue to EOF can be accounted for by a corresponding change in the filmthickness h. However, this is somewhat a cosmetic step as verticaltransport is not regarded as a limiting factor.

More importantly, it is necessary to account for changes in amount ofanalyte in the film and for concentration changes. For want of a moreaccurate alternative in depicting the real system, it is assumed thatthe analyte that is injected into the capillary is reflected by a changein analyte concentration throughout the film, in a manner proportionalto the analyte concentration in each zone:

    C.sub.i,k (new)=C.sub.i,k (old)·(m.sub.i,rem -Q.sub.eo ΔtC.sub.i,1 /m.sub.i,rem ·(h/(h-Δh)) (28)

where mi,rem is the residual amount of analyte i in the film, and Δh isgiven by:

    Δh=Q.sub.eo Δt/(πr.sub.out.sup.2)           (30)

where h is the current thickness of the film. Note that on the righthand side of equation (28) the first term accounts for change in theamount, while the second term accounts for change in volume by changingthe thickness of the film.

In applicants' experimental system, EOF was very slow and Q_(eo)accounts for only 1.5% of the film volume over a 30 s EI period at 3 kV.As such, this refinement of applicants' model leads to hardly anynoticeable change, e.g., for a 10 s EI period with acetate, the fractionintroduced increases from 91.11% to 91.33%. However, a greater change isobserved at higher EOF, e.g., using the same example as above, thefraction introduced was 93.42% when the EOF was tenfold greater.

As noted, it is desired to maintain a finite, small microreservoir,preferably less than about 1 μL in volume. Applicants' wire loop, asdepicted in FIGS. 3A, 3B, advantageously provide such a microreservoir.Further, such a microreservoir also provides the ability to subject theentire sample within to a uniform electric field, by using themicroreservoir metal loop as a high voltage electrode. Othermicroreservoir configurations have been examined by applicants, e.g., aconductive hemisphere such as was described with respect to FIG. 3C.

Consider the effects of the radius dimension of a wire loop or ahemispheric microreservoir. Understandably, the sample volumeeffectively changes when the radius is changed. For a given appliedvoltage, a considerably longer time period is required for "exhaustive"EI. FIG. 9 depicts the results of a numerical simulation for fractionsof chloride and acetate injected as a function of microreservoir loopradius and EI period.

For reference purposes, applicants performed and examined conventionalhydrodynamic and conventional EI injection from a vial, using the samesystem and loop-electrode as has been described herein. Hydrodynamicinjection was linear with time, e.g., for 10 s to 90 s, the heightdifference was 4.7 cm, using N,N-dimethylformamide as sample.

The peak area in arbitrary units is given by equation (27) as follows:

    peak area(arb. units)=12.39±0.37t(sec)-(19.66±3.78);

    r.sup.2 =0.9867                                            (27)

Conventional EI was carried out from a vial with a Cl⁻, NO₃ ⁻, HCOO⁻,CH₃ COO⁻ trace standard mixture. For an EI period of 1 s to 30 s with aHV of -3 kV, all of the individual ions were introduced at a ratelinearly proportional to the EI period. Individual r² values were 0.9976(Cl⁻), 0.9980 (NO₃ ⁻), 0.9968 (HCOO⁻), and 0.9904 (CH₃ COO⁻). Neitherthe concentration of the individual analytes in the sample, nor theirresponse factors were the same. Thus, it is convenient to express theresults in terms of the equivalent volumetric sample introduction rate.This rate varies with the mobility of the sample ion, being 0.489±0.008μL/s (Cl⁻), 0.443±0.007 μL/s (NO₃ ⁻), 0.338±0.007 μL/s (HCOO⁻), and0.2373±0.008 μL/s (CH₃ COO⁻) μL/s.

The relation of the injection rate dV_(inj) /dt with ionic mobilityμ_(i) is given by equation (28) as:

    (dV.sub.inj /di)=(6.69±0.24)·102 μ.sub.i -(4.51±1.54)·10.sup.-2,

    r.sup.2 =0.9975                                            (28)

The above result is in close agreement with what equation (1) wouldpredict. However, these results may be the best that can be expectedfrom such systems, and marked variations from the behavior expected fromequation (1) are observable at higher applied voltages.

Applicants conducted experiments at applied EI voltages of -6 kV and upto 15 s, and -9 kV and up to 10 s, in addition to the above described -3kV measurements. At each applied voltage, the relationship between thesample amount introduced and the EI period was linear for Cl⁻ and NO₃(r² >0.9970). However, for HCOO⁻ the r² value was 0.9829 at -9 kV, andfor CH₃ COO the linear r² value degraded to 0.96 at -6 kV and to 0.25 at-9 kV.

More significantly, based upon equation (1), one would expect thatsample introduction rates would be linearly proportional to the appliedvoltages. For Cl⁻ and NO₃ ⁻, the ratio of the introduction rates at -3kV, -6 kV, and -9 kV were 1:1.59:1.72 and 1:1.45:1.50, respectively,instead of the expected result 1:2:3. Indeed, the introduction rate ofnitrate hardly varied between -6 kV and -9 kV. For formate, the observedratio as a function of the applied voltage was 1:1.25:0.98 and theintroduction rate actually decreased at -9 kV. This effect was morepronounced for acetate, with an observed ratio of 1:0.83:0.05. At thehighest applied voltage, very little acetate was introduced at all, andthe amount had very little dependence on the EI period (viz., low r²cited above).

It is unlikely that electrochemical reduction of acetate is a plausiblemechanism to account for this behavior. Applicants instead presume thatthis reduction originates in depletion near the capillary tip of theslow moving ions. If so, decreasing the concentration of the analyteswould worsen the situation, especially for the more susceptible ions.

FIGS. 10A and 10B depict superimposed electropherograms of applicants'trace test standard mixture (FIG. 10A-(a)), and the solution of FIG.10A-(a), but diluted four times (FIG. 10A-(b)), both solutions subjectedto EI for 30 s at -3 kV. Note that formate and acetate are not evendiscernible in the pherogram of FIG. 10A-(b).

In general, such behavior is at least qualitatively understandable. Whena capillary and a HV electrode are dipped into a sample vial and EI isattempted, the electric field is present primarily between the capillarytip and the electrode. Thus, effectively only a small part of the samplevolume is subjected to the electric field. As EI progresses, theelectrolytic production of acid or base further concentrates theelectrical field to the same region due to increasing conductance.

In a loop, the hypothetical successive radial zones are electricallyeffectively in series with each other. But in conventional EI, thevarious pathways between the capillary tip and the electrode areeffectively in parallel, not in series. The major mechanism ofreplenishment of the analyte ions in this primary field region isdiffusion. For an ion of small diffusion coefficient, little transferoccurs. As a result, no concentration gradient develops within the bulksolution, which further inhibits diffusive transport, aself-perpetuating effect. Obviously, the bias will increase if theanalyte concentration is low to start with, or if the applied electricfield is high.

Consider now electromigration injection (EI) from a symmetricalmicroreservoir-electrode, such as applicants' loop, and the role ofmobility-based bias. An especially important aspect of EI from a loop isthat the type of bias depicts in FIG. 10A-(a) and FIG. 10A-(b) isneither theoretically expected, nor experimentally observed. FIG.10B-(c) depicts the same conditions as in FIG. 10A-(b), except that EIis now performed (at the same voltage for the same period) from a loopof 1 μL volume. Not only are all the four expected peaks observed, but apeak due to carbonate is also observed.

A terminating electrolyte such as sodium pentanesulfonate is often addedin conventional EI to improve trace analysis, and this was done for thedata of FIG. 10B-(d). It is apparent from FIG. 10B that nothing isgained in EI from a loop from the addition of such electrolyte. Theorysuggests that such bias encountered in conventional EI will beassociated with the geometry of the field. Accordingly, applicantsconducted an experiment in which the sample was contained in ahemispherical depression of about 10 μL volume in a metal block thatalso functioned as the HV electrode (see FIG. 3C). The capillary tip waslocated at the center of this depression. The results are quite similarto that obtained with the loop, underscoring the importance of thegeometry of the field experienced by the sample.

FIGS. 11A and 11B depict an even more striking example of the dynamicrange of EI from a loop. In FIG. 11A, acetate was varied between 30 ppbto 30 ppm while nitrate was held constant at 300 ppb. For the data inFIG. 11B, acetate was held constant at 200 ppb and nitrate was similarlyvaried. The peak areas, corrected for migration time shift, demonstratethat the technique disclosed herein by applicants advantageouslyprovides substantially greater dynamic range than is possible withconventional EI.

The results of sample introduction by EI from a loop were investigatedat applied voltages ranging from 3 kV to 18 kV, varied in 3 kV steps,for V·t products ranging from 9 kV·s to 108 kV·s. The loop-EI resultsare quite different from vial-EI, as described earlier herein. For agiven V·t product, introduction is more complete at higher appliedvoltage ,as theory predicts.

FIG. 12 depicts change in chloride/acetate peak area ratio as a functionof the EI period at different applied voltages for a 1 μL loop. For thisplot, the area ratio when the highest voltage (18 kV) has been appliedfor the smallest period of time (0.5 s), has been arbitrarily assignedthe value of unity. While the bias would be the greatest under theseconditions, it is expected to approach the limiting value of 0.535(acetate/chloride mobility ratio) at higher V·t products. Indeed, thisis observed.

The completeness of injection and amount of bias was investigated. FIGS.13A and 13B depict data from an approximately 1 μL volume inclined loopof about 1 mm radius (see FIG. 3B). Because the exact volume wasdifficult to determine, FIG. 13 uses an ordinate of equivalent volumeinjected, i.e., analyte contained within that volume. The results arequalitatively similar to the model calculations described in associationwith presented in FIG. 5. However efficiency was less, in that about 50s time was required to accomplish what required 10 s in the earliermodel. Applicants believe this is a consequence of the fact that thepresent model does not take into account limitations in verticaltransport, e.g., a 320 μm thick film is hardly infinitesimally thin.However the broad general pattern is the same as that in the model. FIG.13B depicts that with exhaustive electromigration, bias is greatlydiminished, although it is not completely eliminated.

As may be theoretically expected, applicants have observed that largerloops lead to less complete injection and greater bias when EI isconducted for the same period at the same voltage. On the other hand,because vertical planar loops hold less liquid, injection is morecomplete and bias is less with these loops under the same EI conditionsfor the same loop radius. Since hydroxide is formed as the result of EI,it is injected into the system. The BGE has a finite buffer capacity andthe OH⁻ peak thus appears only after this capacity is exceeded. Anexample is shown in FIG. 14, in which no hydroxide peak is visible for a15 s EI period, but is by far the major peak by an EI period of 25 s.The integrated hydroxide peak area for EI periods ranging from 25 s to60 s is correlated linearly with the EI period with an r² value of0.9996. A linear relationship of OH⁻ introduction with time was seen inFIG. 4. It is also predictable that a positive peak due to hydroxidefirst occurs after 20 s of EI.

In summary, applicants have demonstrated that substantial improvementsin EI in CE can result if injection is made from a small finite volumein which the entire sample is exposed to the electrical field,preferably symmetrically. Indeed, the use of microreservoir-electrodesdisclosed herein may constitute a more convenient and powerful injectionmethod in CE than what is presently practiced in the prior art.Understandably, applicants' results using films formed on wire loops maybe applied to other geometries as well, in addition to forminghemispherical wells in a microtiter metallized plate. An autosamplerconfiguration may be used with a variety of microreservoir-electrodes.

Applicants anticipate that the present invention can not only improvesampling and injection in CE and other separation systems, but willsimultaneously improve detection limits beyond what is presentlyavailable with conventional detectors. Introduction of H⁺ or OH⁻presumably can be prevented using membrane arrangements, should suchintroduction become a significant factor. Modifications and variationsmay be made to the disclosed embodiments without departing from thesubject and spirit of the invention as defined by the following claims.For example, although the present invention has been described primarilywith respect to tests made using a capillary electrophoresis separationsystem, the present invention may also be practiced in a capillaryelectrochromatography separation system.

What is claimed is:
 1. In a capillary separation system in which analyteions of a chosen polarity in a sample are caused to migrate through aseparation capillary by application of high voltage, a method ofexhaustively injecting said analyte ions by electromigration into saidcapillary such that injection is substantially independent of ionicmobility of individual ones of said analyte ions and is substantiallyindependent of sample conductivity, the method comprising the followingsteps:(a) providing an electrically conductive symmetrically-shapedmicroreservoir fabricated from a conductive wire formed into a circularloop to define a desired loop area that defines a substantially constantfinite holding volume in which a reproducible volume of said sample isretained; (b) disposing an entry tip of said separation capillary incontact with a portion of said sample in said microreservoir; and (c)subjecting said sample in said microreservoir to a radially symmetricelectric field by coupling one lead of said high voltage to saidmicroreservoir and energizing said high voltage for a time periodcausing ions in said sample of said chosen polarity to be substantiallyexhaustively introduced into said tip of said separation capillary. 2.The method of claim 1, wherein said system is an electrophoreticseparation system.
 3. The method of claim 1, wherein said system is acapillary electrochromatographic separation system.
 4. The method ofclaim 1, wherein step (a) includes fabricating said microreservoir witha loop plane oriented in a plane parallel to a longitudinal axis of saidseparation capillary.
 5. The method of claim 1, wherein step (a)includes fabricating said microreservoir with a loop plane offset from alongitudinal axis of said separation capillary by an angle ranging fromabout 45° to about 90°.
 6. The method of claim 1, wherein saidmicroreservoir defines a holding volume that is symmetrically shaped. 7.The method of claim 1, wherein step (b) includes disposing said entrytip of said separation capillary in contact with a portion of saidsample at a symmetrically central portion of said microreservoir.
 8. Themethod of claim 1, wherein at step (a) said holding volume is a volumeselected from the group consisting of (a) a volume less than about 2 μL,(b) a volume less than about 1 μL, and (c) a volume less than about 0.5μL.
 9. In a capillary separation system in which analyte ions of achosen polarity in a sample are caused to migrate through a separationcapillary by application of high voltage, a method of exhaustivelyinjecting said analyte ions by electromigration into said capillary suchthat injection is substantially independent of ionic mobility ofindividual ones of said analyte ions and is substantially independent ofsample conductivity, the method comprising the following steps:(a)providing an electrically conductive symmetrically-shaped microreservoirthat includes a base portion in which there is defined a symmetricalhemisphere cavity having a desired constant finite holding volume inwhich a reproducible volume of said sample is retained; (b) disposing anentry tip of said separation capillary in contact with a portion of saidsample in said microreservoir; and (c) subjecting said sample in saidmicroreservoir to a radially symmetric electric field by coupling onelead of said high voltage to said microreservoir and energizing saidhigh voltage for a time period causing ions in said sample of saidchosen polarity to be substantially exhaustively introduced into saidtip of said separation capillary.
 10. The method of claim 9, whereinsaid system is an electrophoretic separation system.
 11. The method ofclaim 9, wherein said system is a capillary electrochromatographicseparation system.
 12. The method of claim 9, wherein step (b) includesdisposing said entry tip of said separation capillary in contact with aportion of said sample at a symmetrically central portion of saidmicroreservoir.
 13. The method of claim 9, wherein at step (a) saidholding volume is a volume selected from the group consisting of (a) avolume less than about 2 μL, (b) a volume less than about 1 μL, and (c)a volume less than about 0.5 μL.